In central synapses, synaptobrevin2 (syb2, also called VAMP2) is the predominant synaptic vesicle SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein that interacts with the plasma membrane SNAREs SNAP-25 and syntaxin1 to execute exocytosis. However, while neurons lacking syb2 have a nearly complete absence of evoked neurotransmission, they still maintain significant levels of spontaneous neurotransmitter release. The physiological role of this residual spontaneous release after genetic deletion of syb2 has remained elusive. Recent studies have shown that alternative vesicular SNARE proteins such as VAMP4, VAMP7 (also called tetanus-insensitive or TI-VAMP) and Vps10p tail interactor 1 a (Vti1a) functionally diverge from syb2 and independently carry out spontaneous and some asynchronous neurotransmitter release. These studies demonstrated that these alternative vesicular SNAREs constitute molecular tags for independently functioning synaptic vesicle populations and provide a potential molecular basis for selective regulation of distinct forms of neurotransmitter release. In this application, we propose to examine the physiological impact of the forms of neurotransmitter release mediated by these alternative SNAREs. We will delineate how neurotransmitter release mediated by these alternative SNAREs directs neuronal signaling and synaptic efficacy via three Specific Aims. In the first aim, the synaptic scaling elicited by selective manipulation of spontaneous neurotransmitter release will be examined. The second aim will focus on the postsynaptic Ca2+ signals elicited by spontaneous neurotransmitter release. Finally, the third aim will investigate the regulation of synaptic plasticity by selective manipulation of spontaneous neurotransmitter release in an intact synaptic circuit. Collectively, these complementary experiments will elucidate how spontaneous neurotransmission modulates neuronal function in a physiological network. Information attained from these studies will provide new insight to the synaptic substrates that may be affected by a number of in neuropsychiatric and neurological disorders including major depressive disorder, autism and schizophrenia.